1. Field of the Invention
The invention relates generally to agricultural biotechnology. In particular, the invention relates to polynucleotides isolated from coffee plants that are expressed predominantly during the late stages of grain development and maturation. It also relates to promoters that regulate expression of these polynucleotides in plants and methods for their use.
2. Description of the Related Art
The development and maturation of seeds, including those of coffee (grain/bean), follows a specific, tightly-regulated developmental pathway that is driven by temporally-distinct changes in gene and protein expression (Girke, T. et al., 2000; Fait, A. et al., 2006; Hajduch, M. et al., 2005; De Castro, R. D. and Marraccini, P., 2006)). Any genetic modification of a seed requires that the added “modified” transgene(s) be controlled by a promoter sequence capable of “driving” expression in the seed. To limit the “interference” of the introduced transgene on other parts of the plant, it is often desirable that the promoter used to drive the transgene's expression functions only at a specific times, e.g., under particular conditions or during a particular stage of seed development.
There are currently several coffee DNA promoter sequences available. Some are capable of driving strong expression of transgenes in green tissue like leaves, and potentially very weak expression in immature grain such as the rbcS promoter (Marraccini, P. and Rogers J., 2006). Leaf-specific gene promoters from coffee are capable of directing strong expression primarily in the coffee seed during endosperm formation and expansion, see for example U.S. Pat. No. 7,153,953; also 11S promoter (Marraccini, P. and Rogers J., 2003), coffee storage protein promoter (U.S. Pat. No. 6,617,433); oleosin promoter (WO 2007/005928, Simkin, A. J. et al., 2006b), an dehydrin promoter (WO 2007/005980, Tanksley, S. et al., 2007) and (Hinniger, C. et al., 2006).
However, there are currently no coffee promoters available that control strong expression of recombinant genes, exclusively, or nearly exclusively, during late coffee grain maturation. That stage is the maturation stage, involving partial seed dehydration. There are very few well-characterized late grain-specific promoters described in the literature for any seeds. It is considered important to have such genetic elements, to better understand seed maturation, including of coffee grain. Such promoters would have many uses, including improving the quality of coffee in the consumer's cup. These promoters would also allow experimental work, such as testing the effect of expressing recombinant genes at late stages of seed development.
For example, in attempts to improve the quality or extractability of coffee, it is possible that expressing candidate genes during endosperm development/expansion could cause defects in the grain. Expressing the same candidate genes later in development, however, may have no effect on the grain structure/function. Because it is important to test the effects of expressing candidate “coffee quality” gene sequences during the late stages of development there is a need for late grain development promoters.
In the field, coffee maturity is generally considered to be represented by red cherries, and/or softening of the coffee cherry fruit (pericarp). However, there is little detailed experimental evidence indicating that the grain of all fully red cherries have reached complete maturity in all the different commercial varieties. In fact, it is conceivable that the “red” color development of the cherries can, at least in a few varieties, occur faster than the final development of the internal coffee grain. Furthermore, although ideally all coffee is harvested at the red cherry stage, there is a significant level of harvesting, including mechanical harvesting of C. arabica, which includes less ripe yellow and sometimes even green/yellow cherries. Therefore, lots or batches of harvested coffee can contain certain levels of immature grain. In addition, unscrupulous traders have been known to mix batches of immature and mature cherries to prepare coffee lots that are “acceptable” to buyers because “immaturity-related” defects are less evident.
The presence of such immature beans can result in a major defect of green coffee ((Farah, A. and Donangelo, C. M., 2006) and references therein). The lower flavour quality associated with immature beans is likely to be due to multiple factors, including differences in the grain structure before and after full maturation. Certain components present in the immature grain may be potentially detrimental to the flavour quality when immature beans are present in lots of harvested coffee. For example, lower levels of sucrose (Geromel, C. et al., 2006; Privat, I. et al., 2006), nucleic acids and proteins associated with sucrose accumulation in coffee (WO 2007/022318 A2) and higher levels of chlorogenic acids (Farah, A. and Donangelo, C. M., 2006) may all adversely impact coffee flavour and quality.
As indicated above, the presence of immature grain, whether through lack of quality assurance, lack of adequate harvesting practices, or lack of ethics (e.g., intentional deception), results in suboptimal or inferior coffee and is problematic for buyers of quality or premium coffees. There are currently no tests available to measure the maturity level of stored grains, such as coffee grains, particularly where the harvesting practices, mixing, or storage conditions are either not known, or not under the direct control of the buyer. There is a need for methods and techniques capable of detecting the presence of immature grain in lots of coffee in agricultural or industrial settings. Such methods would be useful, for example, within the coffee trade, where the results could reflect the maturity of the lot, and thus determine the quality, grading, acceptability, or value of the lot. Such methods would also be useful for determining the properties of other valuable grains or seeds including corn, soya, etc.).
There remains a need, therefore, for genes and promoters from plants that are expressed predominantly during the late stages of grain development and maturation. In particular, there is a need for such polynucleotides from coffee, as well as methods for the use of these polypeptides in assessing the quality of grains used as agricultural commodities.